Anti-inflammatory compounds in combination with hydrogen for the treatment of inflammation

ABSTRACT

A composition for treating inflammation and pain, including a hydrogen-generating compound in an amount that increases the amount of hydrogen in an individual and has an anti-inflammatory effect. A composition for treating inflammation and pain, including synergistically effective amounts of pregabalin and lactulose. A method of treating inflammation and pain, by administering a composition comprising a hydrogen-generating compound to an individual in an amount that increases the amount of hydrogen in the individual and has an anti-inflammatory effect.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to compositions and methods for treatinginflammation. More specifically, the present invention relates toanti-inflammatory compounds in combination with hydrogen andhydrogen-generating compounds for treating inflammation.

2. Background Art

There are currently many anti-inflammatory/anti-pain agents that areused to treat inflammation and pain in patients. These agents generallyinclude steroids and non-steroidal anti-inflammatory drugs (NSAIDS).Steroids generally act to reduce inflammation by binding to theglucocorticoid receptor, whereas NSAIDS generally act to inhibit bothcyclooxygenase-1 (COX-1) and cyclooxygenase (COX-2), thus inhibiting thecatalysis of the formation of the inflammation messengers prostaglandinsand thromboxane.

These anti-inflammatory/anti-pain agents are widely used but can havemany adverse side effects. Steroids have been shown to causehyperglycemia, insulin resistance, diabetes, osteoporosis, cataracts,anxiety, depression, colitis, hypertension, ictus, erectile dysfunction,hypogonadism, hypothyroidism, amenorrhea, retinopathy, and teratogenicdefects. NSAIDS have been shown to cause gastrointestinal adversereactions (nausea, dyspepsia, gastric ulceration and bleeding,diarrhea), myocardial infarction, stroke, erectile dysfunction, renaladverse reactions (salt and fluid retention, hypertension, interstitialnephritis, nephrotic syndrome, acute renal failure, acute tubularnecrosis), photosensitivity, teratogenic defects, premature birth,miscarriage, raised liver enzymes, headache, dizziness, hyperalaemia,confusion, bronchospasm, rashes, swelling, and irritable bowel syndrome.

One particular anti-inflammation and anti-pain agent that has previouslybeen widely used is gabapentin (NEURONTIN®, Pfizer, Inc.). Gabapentin isa GABA analogue and is indicated for controlling seizures as well as forrelieving neuropathic pain. Pain relief is brought about with gabapentinby changing the way in which the body senses pain. More specifically,gabapentin prevents excessive electrical activity in the brain bymimicking the activity of the neurotransmitter GABA. GABA is a naturalnerve calming agent, and by mimicking this action, gabapentin can calmnerve activity in the brain.

The successor drug developed to gabapentin was pregabalin (LYRICA®,Pfizer, Inc.). Pregabalin is also a GABA analogue, and is theS-enantiomer of 3-aminomethyl-5-methyl-hexanoic acid. Pregabalin isindicated for neuropathic pain and seizures, and is more specificallyindicated for chronic pain disorders such as fibromyalgia. Pregabalinhas been preferred over gabapentin due to the fact that it can providethe same efficacy but at much lower doses than are required forgabapentin. Equivalent efficacy at a lower dose can be achieved withpregabalin due to the fact that it has a higher bioavailability and israpidly absorbed by the body in comparison to gabapentin. Therefore,administering pregabalin can overcome some adverse side effects relatedto dosing with gabapentin. However, adverse effects can still remainwhen taking pregabalin, such as commonly reported dizziness ordrowsiness, as well as withdrawal effects and other effects as describedabove.

Therefore, there remains a need for an anti-inflammatory/anti-pain agentthat is effective in treating inflammation and pain but reduces the riskof the above adverse reactions.

SUMMARY OF THE INVENTION

The present invention provides for a composition for treatinginflammation and pain, including a hydrogen-generating compound in anamount that increases the amount of hydrogen in an individual and has ananti-inflammatory effect.

The present invention also provides for a composition for treatinginflammation and pain, including synergistically effective amounts ofpregabalin and lactulose.

The present invention provides for a method of treating inflammation andpain, by administering a composition comprising a hydrogen-generatingcompound to an individual in an amount that increases the amount ofhydrogen in the individual and has an anti-inflammatory effect.

DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention are readily appreciated as thesame becomes better understood by reference to the following detaileddescription when considered in connection with the accompanying drawingswherein:

FIG. 1A is a photograph of a foot pad at day 0, and FIG. 1B is aphotograph of foot pads at day 8 of administration of the composition ofthe present invention;

FIG. 2 is a graph of foot pad thickness on day 7;

FIG. 3 is a graph of H2 concentration in the abdomen on day 8;

FIG. 4 is a graph of NGF in the foot pad;

FIG. 5 is a graph of substance P (pain marker);

FIG. 6 is a graph of levels of IL-1β in tissue;

FIG. 7 is a graph of H2 concentration in ascites fluid after ingestionof sugar solution;

FIG. 8 is a graph of H2 concentration in ascites fluid after ingestionof H2-enriched water versus lactulose;

FIG. 9A is a depiction of inulin and FIG. 9B is a depiction oflactulose;

FIG. 10 is a graph of H2 attenuated Celecoxib-mediated promotion ofTNF-α production from LPS-stimulated mouse macrophages;

FIG. 11 is a graph of the effects of H2 on ibuprofen-mediated TNF-αsuppression;

FIG. 12 is a graph of mouse FCA induced foot pad inflammation;

FIG. 13 is a graph of the measurement of PGE2 in inflamed foot pads;

FIG. 14 is a graph of the measurement of NGF in inflamed foot pads; and

FIG. 15 is a graph of the measurement of TNF-α in inflamed foot pads.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is generally directed to compositions and methodsthat are useful for treating inflammation and/or pain. Morespecifically, the present invention is directed to a composition of ananti-inflammatory or anti-pain agent in combination with a compound thatgenerates increased hydrogen in the body that can be used to treatinflammation and pain.

“Hydrogen” as used herein, refers to the composition H₂ (also writtenherein as “H2”), but can also include molecular hydrogen (H) and anycomposition capable of releasing hydrogen. In other words, H2 moleculesper se can be administered, a prodrug able to release H2, or a compoundthat can cause the release of H2 within the body can be administered, asfurther described below.

The anti-inflammatory or anti-pain agent can be any agent that reducesinflammation and/or treats pain. It should be noted that many of theseagents both reduce inflammation and reduce pain; however, some agentscan perform only one of these functions as well.

The anti-inflammatory/anti-pain agent can be non-steroidalanti-inflammatory drugs (NSAIDS) such as, but not limited to,acetaminophen, salicylates (aspirin, diflunisal, salsalate), acetic acidderivatives (indomethacin, ketorolac, sulindac etodolac, diclofenac,nabumetone), propionic acid derivatives (ibuprofen, naproxen,flurbiprofen, ketoprofen, oxaprozin, fenoprofen, loxoprofen), fenamicacid derivatives (meclofenamic acid, mefenamic acid, flufenamic acid,tolfenamic acid), oxicam (enolic acid) derivatives (piroxicam,meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam), arylalkanoic acidderivatives (tolmetin); or selective COX-2 inhibitors (celecoxib,rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib, firocoxib).The anti-inflammatory/anti-pain agent can also be steroids such as, butnot limited to, corticosteroids (hydrocortisone, hydrocortisone acetate,cortisone acetate, tixocortol pivalate, prednisolone,methylprednisolone, prednisone, triamcinolone acetonide, triamcinolonealcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide,fluocinolone acetonide, halcinonide, betamethasone, dexamethasone,fluocortolone, hydrocortisone-17-valerate, aclometasone dipropionate,betamethasone valerate, betamethasone dipropionate, prednicarbate,clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolonecaproate, fluocortolone pivalate, or fluprednidene acetate). Theanti-inflammatory/anti-pain agent can further be immune selectiveanti-inflammatory derivatives (ImSAIDs) such as, but not limited to,submandibular gland peptide T (SGp-T) and derivativesphenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG).

Ibuprofen (ADVIL®, Pfizer) is an NSAID that can be used in combinationwith the hydrogen-generating compound in the present invention.Ibuprofen is generally known as(RS)-2-(4-(2-methylpropyl)phenyl)propanoic acid.

Ibuprofen can be administered in oral form in doses of 200 to 3200mg/day; however, due to synergism with the hydrogen-generating compound,a lower dose can be preferred and normal side effects experienced can bereduced or eliminated. Any other administration methods as describedherein can also be used. Administration of Ibuprofen in combination withH2 or lactulose is shown to reduce inflammation in a greater amount thanadministration of Ibuprofen alone, as shown in Examples 3 and 4 below.

Celecoxib (CELEBREX®, GD Searle) is another particular NSAID that can beused in combination with the hydrogen-generating compound in the presentinvention. Celecoxib is generally described in U.S. Pat. No. 5,466,823to Talley, et al. Briefly, celecoxib is a compound that is useful intreating inflammation-related disorders defined by Formula I:

wherein R¹ is selected from sulfamyl, halo, alkyl, alkoxy, hydroxyl andhaloalkyl; wherein R² is selected from hydrido, halo, haloalkyl, cyano,nitro, formyl, carboxyl, alkoxycarbonyl, carboxyalkyl,alkoxycarbonylalkyl, amidino, cyanoamidino, amido, alkoxy, amidoalkyl,N-monoalkylamido, N-monoarylamido, N,N-dialkylamido,N-alkyl-N-arylamido, alkylcarbonyl, alkylcarbonylalkyl, hydroxyalkyl,alkylthio, alkylsulfinyl, alkylsulfonyl, N-alkylsulfamyl,N-arylsulfamyl, arylsulfonyl, N,N-dialkylsulfamyl,N-alkyl-N-arylsulfamyl and heterocyclic; wherein R³ is selected fromhydrido, halo, haloalkyl, cyano, nitro, formyl, carboxyl,alkoxycarbonyl, carboxyalkyl, alkoxycarbonylalkyl, amidino,cyanoamidino, amido, alkoxy, amidoalkyl, N-monoalkylamido,N-monoarylamido, N,N-dialkylamido, N-alkyl-N-arylamido, alkylcarbonyl,alkylcarbonylalkyl, hydroxyalkyl, alkylthio, alkylsulfinyl,alkylsulfonyl, N-alkylsulfamyl, N-arylsulfamyl, arylsulfonyl,N,N-dialkylsulfamyl, N-alkyl-N-arylsulfamyl, heterocyclic,heterocycloalkyl and aralkyl; wherein R⁴ is selected from aryl,cycloalkyl, cycloalkenyl and heterocyclic; wherein R⁴ is optionallysubstituted at a substitutable position with one or more radicalsselected from halo, alkylthio, alkylsulfinyl, alkyl, alkylsulfonyl,cyano, carboxyl, alkoxycarbonyl, amido, N-monoalkylamido,N-monoarylamido, N,N-dialkylamido, N-alkyl-N-arylamido, haloalkyl,hydroxyl, alkoxy hydroxyalkyl haloalkoxy, sulfamyl, N-alkylsulfamyl,amino, N-alkylamino, N,N-dialkylamino, heterocyclic, nitro andacylamino; or wherein R³ and R⁴ together form

and m is 1 to 3, inclusive; and wherein R⁵ is one or more radicalsselected from halo, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano,carboxyl, alkoxycarbonyl, amido, N-monoalkylamido, N-monoarylamido,alkyl, N,N-dialkylamido, N-alkyl-N-arylamido, haloalkyl, hydrido,hydroxyl, alkoxy, hydroxyalkyl, haloalkoxy, sulfamyl, N-alkylsulfamyl,amino, alkylamino, heterocyclic, nitro and acylamino; provided R² and R³are not identical radicals selected from hydrido, carboxyl andethoxycarbonyl; further provided that R² cannot be carboxyl when R³ ishydrido and when R⁴ is phenyl; and further provided that R⁴ is sulfamylor N-alkylsulfamyl when R¹ is halo; or a pharmaceutically-acceptablesalt thereof.

Celecoxib can be administered in oral form in doses of 50 to 400 mg/day;however, due to synergism with the hydrogen-generating compound, a lowerdose can be preferred and normal side effects experienced can be reducedor eliminated. Any other administration methods as described herein canalso be used. Administration of celecoxib in combination with H2 orlactulose is shown to reduce inflammation in a greater amount thanadministration of celecoxib alone, as shown in Examples 3 and 4 below.

Rofecoxib (VIOXX®, Merck) is another particular NSAID that can be usedin combination with the hydrogen-generating compound in the presentinvention. Rofecoxib is generally described in U.S. Pat. No. 5,474,995to Ducharme, et al. and U.S. Pat. No. 5,691,374 to Black, et al.Briefly, the rofecoxib compound of Formula II

or pharmaceutically acceptable salts thereof is useful in the treatmentof cyclooxygenase-2 mediated diseases, wherein: X—Y—Z is selected fromthe group consisting of:

-   -   (a) —CH₂CH₂CH₂—,    -   (b) —C(O)CH₂CH₂—,    -   (c) —CH₂CH₂C(O)—,    -   (d) —CR⁵(R⁵)—O—C(O)—,    -   (e) —C(O)—O—CR⁵(R⁵′)—,    -   (f) —CH₂—NR³—CH₂—,    -   (g) —CR⁵(R⁵′)—NR³—C(O)—,    -   (h) —CR⁴═CR⁴′—S—,    -   (i) —S—CR⁴═CR⁴′—,    -   (j) —S—N═CH—,    -   (k) —CH═N—S—,    -   (l) —N═CR⁴—O—,    -   (m) —O—CR4=N—,    -   (n) —N═CR⁴—NH—;    -   (o) —N═CR⁴—S—, and    -   (p) —S—CR⁴—N—;    -   (q) —C(O)—NR³—CR⁵(R⁵)—;    -   (r) —R³N—CH═CH— provided R¹ is not —S(O)₂Me    -   (s) —CH═CH—NR³— provided R¹ is not —S(O)₂Me

when side b is a double bond, and sides a an c are single bonds; and

X—Y—Z— is selected from the group consisting of:

-   -   (a) ═CH—O—CH═, and    -   (b) ═CH—NR³—CH═,    -   (c) ═N—S—CH═,    -   (d) ═CH—S—N═,    -   (e) ═N—O—CH═,    -   (f) ═CH—O—N═,    -   (g) ═N—S—N═,    -   (h) ═N—O—N═,

when sides a and c are double bonds and side b is a single bond;

R¹ is selected from the group consisting of

-   -   (a) S(O)₂CH₃,    -   (b) S(O)₂NH₂,    -   (c) S(O)₂NHC(O)CF₃,    -   (d) S(O)(NH)CH₃,    -   (e) S(O)(NH)NH₂,    -   (f) S(O)(NH)NHC(O)CF₃,    -   (g) P(O)(CH₃)OH, and    -   (h) P(O)(CH₃)NH₂,

R² is selected from the group consisting of

-   -   (a) C₁₋₆ alkyl,    -   (b) C₃, C₄, C₅, C₆, and C₇, cycloalkyl,    -   (c) mono-, di- or tri-substituted phenyl or naphthyl wherein the        substituent is selected from the group consisting of    -   (1) hydrogen,    -   (2) halo,    -   (3) C₁₋₆ alkoxy,    -   (4) C₁₋₆ alkylthio,    -   (5) CN,    -   (6) CF₃,    -   (7) C₁₋₆ alkyl,    -   (8) N₃,    -   (9) —CO₂H,    -   (10) —CO₂—C₁₋₄ alkyl,    -   (11) —C(R⁵)(R⁶)—OH,    -   (12) —C(R⁵)(R⁶)—O—C₁₋₄ alkyl, and    -   (13) —C₁₋₆ alkyl--CO₂—R⁵;    -   (d) mono-, di- or tri-substituted heteroaryl wherein the        heteroaryl is a monocyclic aromatic ting of 5 atoms, said ting        having one hetero atom which is S, O, or N, and optionally 1, 2,        or 3 additionally N atoms; or

the heteroaryl is a monocyclic ring of 6 atoms, said ring having onehetero atom which is N, and optionally 1, 2, 3, or 4 additional N atoms;said substituents are selected from the group consisting of

-   -   (1) hydrogen,    -   (2) halo, including fluoro, chloro, bromo and iodo,    -   (3) C₁₋₆ alkyl,    -   (4) C₁₋₆ alkoxy,    -   (5) C₁₋₆ alkylthio,    -   (6) CN,    -   (7) CF₃,    -   (8) N₃,    -   (9) —C(R⁵)(R⁶)—OH, and    -   (10) —C(R⁵)(R⁶)—O—C₁₋₄ alkyl;    -   (e) benzoheteroaryl which includes the benzo fused analogs of        (d);

R³ is selected from the group consisting of

-   -   (a) hydrogen,    -   (b) CF₃,    -   (c) CN,    -   (d) C₁₋₆ alkyl,    -   (e) hydroxy C₁₋₆ alkyl,    -   (f) —C(O)—C₁₋₆ alkyl,    -   (g) optionally substituted    -   (1) —C₁₋₆ alkyl--Q,    -   (2) —C₁₋₃ alkyl--O—C₁₋₃ alkyl--Q,    -   (3) —C₁₋₃ alkyl--S—C₁₋₃ alkyl--Q,    -   (4) —C₁₋₅ alkyl--O--Q, or    -   (5) —C₁₋₅ alkyl--S--Q,

wherein the substituent resides on the alkyl and the substituent is C₁₋₃alkyl;

-   -   (h) --Q

R⁴ and R^(4′) are each independently selected from the group consistingof

-   -   (a) hydrogen,    -   (b) CF₃,    -   (c) CN,    -   (d) C₁₋₆ alkyl,    -   (e) --Q,    -   (f) —O--Q;    -   (g) —S--Q, and    -   (h) optionally substituted    -   (1) —C₁₋₆ alkyl--Q,    -   (2) —O—C₁₋₅ alkyl--Q,    -   (3) —S—C₁₋₅ alkyl--Q,    -   (4) —C₁₋₃ alkyl--O—C₁₋₃ alkyl--Q,    -   (5) —C₁₋₃ alkyl--S—C₁₋₃ alkyl--Q,    -   (6) —C₁₋₆ alkyl--O--Q,    -   (7) —C₁₋₆ alkyl--S--Q,

wherein the substituent resides on the alkyl and the substituent is C₁₋₃alkyl, and

R⁵, R^(5′), R⁶, R⁷ and R⁸ are each independently selected from the groupconsisting of

-   -   (a) hydrogen,    -   (b) C₁₋₆ alkyl,

or R⁵ and R⁶ or R⁷ and R⁸ together with the carbon to which they areattached form a saturated monocyclic carbon ring of 3, 4, 5, 6 or 7atoms;

Q is CO₂H, CO₂—C₁₋₄ alkyl, tetrazolyl-5-yl, C(R7)(R⁸)(OH), orC(R⁷)(R⁸)(O—C₁₋₄ alkyl);

provided that when X—Y—Z is —S—CR⁴═CR^(4′), then R⁴ and R^(4′) are otherthan CF₃.

Rofecoxib can be administered in oral form in doses of 12.5 to 50mg/day; however, due to synergism with the hydrogen-generating compound,a lower dose can be preferred and normal side effects experienced can bereduced or eliminated. Any other administration methods as describedherein can also be used.

The anti-inflammatory/anti-pain agent can also be a narcotic compositionsuch as, but not limited to, buprenorphine, butorphanol, codeine,hydrocodone, hydromorphone, levorphail, meperidine, methadone, morphine,nalbuphine, oxycodone, oxymorphone, pentaxocine, or propoxyphene.

The anti-inflammatory/anti-pain agent can also be other analgesiccompositions such as, but not limited to, tramadol, or capsaicin. Theanti-inflammatory/anti-pain agent can also be a topical anesthetic, suchas, but not limited to, benzocaine, dibucaine, lidocaine, or prilocaine.

The anti-inflammatory/anti-pain agent can also be any suitable biologicagent that reduces inflammation and/or pain, such as etanercept(ENBREL®, Amgen, Inc.). Etanercept reduces the levels ofinflammatory-causing tumor necrosis factor (TNF) in the body and isadministered by injection. Other biologic agents that inhibit IL-1 orTNF, or that effect other biologic pathways, can also be used, such as,but not limited to, adalimumab (HUMIRA®, Abbott), anakinra (KINERET®,Amgen, Inc.), infliximab (REMICADE®, Janssen Biotech, Inc.),certolizumab-pegol (CIMZIA®, UCB, Inc.), and Natalizumab (TYSABRI®,Biogen Idec).

The anti-inflammatory/anti-pain agent can further be any combination ofthe above compositions along with other agents. Some readily availablecombinations of anti-inflammatory/anti-pain agents are as follows:butalbital, acetameniphen, and caffeine; butalbital, aspirin, andcaffeine; butalbital, acetaminophen, caffeine, and codeine; hydrocodoneand ibuprofen; pentazocine and naloxone; acetaminophen and codeine;dihydrocodeine, acetaminophen, and caffeine; hydrocodone andacetaminophen; oxycodone and acetaminophen; pentazocine andacetaminophen; propoxyphene and acetaminophen; aspirin, caffeine, anddihydrocodeine; aspirin and codeine; hydrocodone and aspirin; oxycodoneand aspirin; pentazocine and aspirin; and propoxyphene, aspirin, andcaffeine.

Most preferably, the anti-inflammatory/anti-pain agent is pregabalin(LYRICA®, Pfizer). Pregabalin is currently indicated for the treatmentof neuropathic pain, seizures, fibromyalgia, and generalized anxietydisorder. Pregabalin and the method of making pregabalin are describedin U.S. Pat. No. 5,847,151. Pregabalin can be administered in oral formin doses of 25 to 600 mg/day; however, due to synergism with thehydrogen-generating compound, a lower dose can be preferred. Any otheradministration methods as described herein can also be used.

Briefly, pregabalin is a series of 3-alkyl-4-aminobutyric acid or3-alkyl glutamic acid analogs. Most preferably, pregabalin is theS-enantiomer of 3-(aminomethyl)-5-methyl-hexanoic acid. However, otheranalogs as discussed herein can also be used and salts of any otheranalogs can be used. Illustrative of the alkyl moieties as representedby R₁ and R₁₁ in Formulas III and IV are methyl, ethyl, propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, isopentyl, andneopentyl as well as other alkyl groups. The cycloalkyl groupsrepresented by R₁ and R₁₁ in Formulas III and IV are exemplified bycyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The more preferred compounds of pregabalin of the present invention areof Formula III above wherein R₃ is hydrogen, R₂ is hydrogen, and R₁ isisobutyl. That is, the preferred compound is4-amino-3-(2-methylpropyl)butanoic acid. It has been found that thiscompound is unexpectedly more potent than the other analogs synthesizedand tested in vivo. What is further surprising, is that this preferredcompound has been found to be the least effective one of the analogstested in activating GAD in vitro. Accordingly, it was very unexpectedthat this preferred compound had such a high potency when tested invivo.

The more preferred compounds of pregabalin of the present invention arethe (S)-(+)- and the (R)(−)-4-amino-3-(2-methylpropyl)butanoic acid withthe (S)-(+)-enantiomer being most preferred. The (S)-(+)-enantiomer wasfound to be the most potent compound within the scope of the presentinvention for displacement of tritiated gabapentin, and both the(S)-(+)- and the (R)-(−)-enantiomers showed pronounced stereoselectivityfor both displacement of tritiated gabapentin and for anticonvulsantactivity in vivo.

The pregabalin compounds made in accordance with the present inventioncan form pharmaceutically acceptable salts with both organic andinorganic acids or bases. For example, the acid addition salts of thebasic compounds are prepared either by dissolving the free base inaqueous or aqueous alcohol solution or other suitable solventscontaining the appropriate acid and isolating the salt by evaporatingthe solution. Examples of pharmaceutically acceptable salts arehydrochlorides, hydrobromide, hydrosulfates, etc, as well as sodium,potassium, and magnesium, etc, salts.

The method for the formation of the 3-alkyl-4-aminobutanoic acidsstarting from 2-alkanoic esters is prepared from commercially availablealdehydes and monomethyl malonate by the Knoevenagel reaction, (Kim Y.C., et al, J. Med. Chem. 1965:8509) with the exception of ethyl4,4-dimethyl-2-pentenoate.

More specifically, the following is a procedure that can be generallyapplied to the preparation of all the 3-alkylglutamic acids. Ten gramsof a 3-alkyl-5,5-dicarbethoxy-2-pyrrolidinone was refluxed in 150 mL of49% fuming HBr for 4 hours. After this time, the contents were placed inan evaporator and the volatile constituents were removed in vacuo withthe aid of a hot-water bath. The gummy residue was dissolved in 25 mL ofdistilled water and the water was removed with the aid of theevaporator. This process was repeated once more. The residue wasdissolved in 20 mL of water, and the pH of the solution was adjusted to3.2 with concentrated NH₃ solution. At this point the chain length ofthe individual 3-alkylglutamic acids altered the solubility so thatthose whose side chains were larger precipitated with the ease fromsolution. Precipitation of the alkylglutamic acids with smallersubstituents (methyl, ethyl, and propyl) could be encouraged by coolingon an ice bath or by diluting the aqueous solution with 100 mL ofabsolute ethanol. Precipitation from the water-alcohol mixture iscomplete in 48 hours. Care must be taken to add the ethanol slowly toprevent the precipitation of an amorphous solid that is notcharacteristic of the desired 3-alkylglutamic acids. Samples of theamino acids were purified for analysis by recrystallizing from awater-ethanol mixture. All melted with decomposition. Melting points ofthe decomposed 3-alkylglutamic acids corresponded with those of theirpyroglutamic acids.

Ethyl 4,4-dimethyl-2-pentenoate was prepared from 2,2-dimethylpropanoland ethyl lithioacetate, followed by dehydration of the β-hydroxy esterwith phosphoryl chloride and pyridine.

The Michael addition of nitromethane to α,β-unsaturated compoundsmediated by 1,1,3,3-tetramethylguanidine or1,8-diazabicyclo-[5.4.0]undec-7-ene(DBU) afforded 4-nitroesters in goodyields. More specifically, a mixture of nitromethane (5 mol),α,β-unsaturated ester (1 mol), and tetramethyl-guanidine (0.2 mol) wasstirred at room temperature for 2 to 4 days. (In case of methylacrylate, the ester has to be added at a temperature below 300.) Theprogress of the reaction was followed by IR (disappearance of the C═Cband) and G. L. C. analysis. The reaction mixture was washed with dilutehydrochloric acid and extracted with ether. The organic extract wasdried, the solvent removed at reduced pressure, and the 20 residuedistilled at a pressure of 2 torr. Although the aliphatic nitrocompounds are usually reduced by either high pressure catalytichydrogenation by metal-catalyzed transfer hydrogenation, or by newlyintroduced hydrogenolysis methods with ammonium formate or sodiumborohydride and palladium as catalysts, applicants have found that4-nitrocarboxylic esters can be reduced almost quantitatively to thecorresponding 4-aminocarboxylic esters by hydrogenation using 10%palladium on carbon as catalysts in acetic acid at room temperature andatmospheric pressure. The amino esters produced were subjected to acidhydrolysis to afford the subject inventive compounds in good yields.This procedure provides access to a variety of 3-alkyl-4-aminobutanoicacids as listed in Tables 1 and 2 as examples and thus is advantageousin comparison to methods previously used.

TABLE 1 Activation of GAD by GABA analogs at various concentrationsexpressed in %

2.5 1.0 0.5 0.25 0.1 0.05 R₁, R₂ mM mM mM mM mM mM (R,S)-CH₃, H 239 168142 128 118 107 (R)-CH₃H 327 202 185 135 128 109 (S)-CH₃H 170 118 — 103— — CH₃, CH₃ 174 125 — 109 — — (R,S)-C₂H₅, H 172 128 — 108 — —(R,S)-n-C₃H₇, H 156 112 — 105 — — (R,S)-i-C₃H₇, H 140 108 — 104 — —(R,S)-n-C₄H₉, H 178 117 — 108 — — (R,S)-i-C₄H₉, H 143 113 — 109 — —(R,S)-s-C₄H₉, H 169 119 — 105 — — (R,S)-t-C₄H₉, H 295 174 147 121 117108 (R,S)-neo-C₅H₁₁, H 279 181 — 130 — — (R,S)-i-C₅H₁₁, H 142 118 — 109— — (R,S)-C₆H₁₃, H 125 100 — 100 — — (R,S)-C₆H₅, H 218 129 — 110 — —

2.5 1.0 0.5 0.25 0.1 0.05 R mM mM mM mM mM mM H(R,S) 140 111 — 104 — —H(R) 173 125 — 108 — — H(S) 100 100 — 100 — — CH₃ 143 121 — 109 — — C₆H₅207 151 — 112 — — Sodium Valproate 207 138 124 119 115 105 GABAPENTIN178 145 — 105 — — Activation of GAD by glutamate analogs expressed in %

R 2.5 mM 1.0 mM 0.25 mM CH₃ 212 144 113 C₂H₅ 170 128 113 n-C₃H₇ 153 125108 i-C₃H₇ 144 114 105 n-C₄H₉ 133 117 105 i-C₄H₉ 129 112 106 C₆H₅ 172135 112 Sodium Valproate 207 138 119

TABLE 2 Prevention of tonic extensor seizures in mice followingintravenous administration of 3-substituted GABA derivatives Effect #Ataxia # Dose Time After Protected/# Ataxia/# R (mg/kg) Dose (min)Tested Tested (R,S)—CH₃ 10 120 0/5  0/5  30 120 4/5  0/5  100 120 3/5 0/5  CH₃ 1 120 1/10 0/10 3 120 2/10 0/10 10 120 4/10 0/10 30 120 3/100/10 100 120 3/10(5/10) 1/10 CH₃ 10 120 1/10 1/10 30 120 2/10 0/10 100120 5/10 0/10 t-C₄H₉ 10 120 2/10 0/10 30 120 2/10 0/10 100 120 5/10 0/10C₂H₅ 3 120 1/5  0/5  10 120 1/5  0/5  30 120 2/5  0/5  100 120 5/5  0/5 (CH₃)₂ 30 120 4/5  0/5  100 120 4/5  0/5  n-C₄H₉ 10 120 1/10 0/10 30 1203/10 0/10 100 120 4/10 0/10 s-C₄H₉ 3 120 2/10 0/10 10 120 3/10 0/10 30120 2/10 0/10 i-C₄H₉ 0.3 120 1/10 0/10 0.8 120 3/10 0/10 2.0 120 5/100/10 5.5 120 7/10 0/10 14.4 120 9/10 0/10 n-C₃H₇ 3 120 2/10 0/10 10 1202/10 3/10 100 120 3/10 0/10 i-C₃H₇ 10 120 5/10 1/10 30 120 5/10 0/10 100120 6/10 0/10 C₆H₅ 100 120 0/10 0/10 neo-C₅H₁₁ 10 120 2/10 0/10 30 1204/10 0/10 100 120 4/10 0/10 High-intensity corneal electroshockconsisted of 50 mA, base-to-peak sinusoidal current for 0.2 seconds. Allother data was from low-intensity electroshock, 17 mA base-to-peaksinusoidal current for 0.2 seconds.

Examples of more specific methods of making compounds in accordance withthe present invention are as follows, optionally utilizing the methodsdescribed in detail above. When the starting material is notcommercially available, the synthetic sequence may be initiated with thecorresponding alcohol, which is oxidized to the aldehyde by the methodof Corey E. J., et al, Tetrahedron Lett. 1975:2647-2650.

The chiral compounds of Formulas III and IV are prepared as set forth inthe schematic in Chart I hereof. Although the schematic in Chart Idepicts the chiral synthesis of specific compound(S)-(+)-4-amino-3-(2-methylpropyl)butanoic acid, one skilled in the artcan readily see that the method of synthesis can be applied to anydiastereomeric compound of Formulas III and IV.

In Chart I Ph is phenyl, Bn is benzyl, THF is tetrahydrofuran, LDA islithium diisopropylamide, BH₃SMe₂ is borane dimethyl sulfide complex,TsCl is tosyl chloride, and DMSO is dimethylsulfoxide.

The detailed synthetic procedure is set forth herein below in Example 1.The key introductory literature for this methodology was discussed inEvans' paper, J. Am. Chem. Soc. 1982; 104:1737-9. The metal enolate canbe formed with a lithium or sodium amide base, and subsequentlyalkylated to give a substituted carboxylic acid derivative. Thismethodology was valuable for the enantioselective synthesis of theseα-substituted carboxylic acid derivatives. In this seminal paper, Evansdescribed the preparation of propionic acid derivatives with a series ofsimple alkylating agents. By varying the stereochemistry of the chiralsynthon (the oxazolidinone), he was able to get high stereoselectivity.

Evans has used this chiral auxiliary in other synthetic studies, butnone has been related to 4-amino-3-(2-methylpropyl)butanoic acid whichcontains a β-substituted-γ-amino acid. The methodology as presented byEvans teaches toward α-substitution, and away from β-substitution, andhas not been used in the preparation of this type of unusual amino acid.N-acyloxazolidinones have been used to form chlorotitanium enolates thathave been reacted with Michael adducts such as acrylonitrile, J. Org.Chem. 1991; 56:5750-2. They have been used in the synthesis of therutamycin family of antibiotics, J. Org. Chem. 1990; 55:6260-8 and instereoselective aldol condensations, Org. Synth. 1990; 68:83-91. Chiralα-amino acids were prepared via the oxazolidinone approach. In thissequence, a dibutylboron enolate was brominated and displaced withazide, Tetrahedron Lett. 1987; 28:1123-6. Other syntheses ofβ-hydroxy-α-amino acids were also reported via this chiral auxiliarythrough aldol condensation (Tetrahedron Lett. 1987; 28:39-42; J. Am.Chem. Soc. 1987; 109:7151-7). α,β-Unsaturated N-acyloxazolidinones havealso been used to induce chirality in the Diels-Alder reaction, (J. Am.Chem, Soc. 1988; 110:1238-56. In none of these examples, or others foundin the literature, is this methodology used to prepare (β-substitutedcarboxylic acids or 3-substituted GABA analogs.

The chiral compounds of Formulas III and IV can also be prepared in amanner which is similar to the synthesis depicted in Chart I. In thisembodiment, however, step 8 in Chart I is replaced by an alternatetwo-step procedure (sodium hydroxide is preferred, however, othersolvents known to those of skill in the art which can hydrolyze theazide (8) to intermediate azide (8a) can be employed). Instead ofreducing the azide (8) to the amino acid (9) in Chart I, the alternateprocedure hydrolyzes the azide (8) to give an intermediate azide (8a)which is subsequently reduced (see Chart Ia).

There are two major advantages to hydrolyzing azide (8) to give theintermediate azide (8a) prior to reduction. The first advantage is thatintermediate azide (8a) may be purified by extraction into aqueous base.After the aqueous extract is acidified, intermediate azide (8a) may beextracted into the organic phase and isolated. This allows for apurification of intermediate azide (8a) which does not involvechromatography. The purification of azide (8) requires chromatographywhich is very expensive and often impractical on a large scale.

The second advantage is that intermediate azide (8a) may be reduced toamino acid (9) without added acid. Reduction of azide (8) requiresaddition of acid, e.g., hydrochloric acid in order to obtain amino acid(9). Unfortunately, lactamization of amino acid (9) is promoted by thepresence of acid. Intermediate azide (8a) may be reduced under nearneutral conditions to give amino acid (9), thus minimizing the problemof lactam formation.

In another preferred embodiment, the chiral compounds of Formulas IIIand IV can be prepared as set forth in the Schematic in Chart II hereof.Although the schematic in Chart II depicts the chiral synthesis ofspecific compound (S)-(+)-4-amino-3-(2-methyl-propyl)butanoic acid, oneskilled in the art can readily see that the method of synthesis can beapplied to any diastereomeric compound of Formulas III and IV.

In Chart II Ph is phenyl, and Ts is tosyl.

Another synthetic procedure is similar to the synthesis route depictedin Chart I, however, the procedure of Chart II replaces the benzyl esterin the synthesis route of Chart I with a t-butyl ester. The desiredamino acid (9) and (109) is the same end product in both Charts I andII, respectively. There are several advantages to using the t-butylester rather than the benzyl ester in the synthesis of amino acid (9) or(109). A first advantage relates to the hydrolysis of the chiralauxiliary in step 4 of Chart 1. During the hydrolysis of the chiralauxiliary in this reaction some hydrolysis of the benzyl ester oftenoccurs. Hydrolysis of the t-butyl ester in Chart II has not beenexperienced.

Another advantage relates to the use of alcohol (106) in Chart II overthe use of alcohol (6) in Chart I. A problem with the benzylester-alcohol is the tendency of the benzyl ester-alcohol to undergolactonization as shown below. Although lactonization of the benzyl estercan be avoided under some conditions, the t-butyl ester-alcohol is farless prone to lactonization.

Still another advantage, which was previously discussed with regard tothe synthetic procedure depicted by Chart Ia, is that the t-butylsynthetic route minimizes the problem of lactam formation of the aminoacid end product (109). Instead of reducing azide (108) to amino acid(109) which requires the addition of acid that causes lactamization ofamino acid (109), azide (108) is first hydrolyzed to intermediate azide(108a). Intermediate azide (108a) may be reduced under neutralconditions to give amino acid (109), thus minimizing the problem oflactam formation.

It should also be mentioned that several novel intermediates areproduced by the processes discussed herein. Some of these intermediateswhich are depicted in Charts I, Ia, and II include in the racemate or Ror S enantiomer form:

4-methyl-5-phenyl-2-oxazolidinone,4-methyl-(2-methylpropyl)-2-dioxo-5-phenyl-3-oxazolidine butanoic acid,phenylmethyl ester, 4-methyl-pentanoyl chloride,4-methyl-3-(4-methyl-1-oxopentyl)-5-phenyl-2-oxazolidinone,2-(2-methylpropyl)-butanedioic acid, 4-(phenylmethyl)ester,3-(azidomethyl)-5-methyl-hexanoic acid, phenylmethyl ester,3-(hydroxymethyl)-5-methyl-hexanoic acid, phenylmethyl ester,5-methyl-3-[[[(4-methylphenyl)sulfonyl]oxy]-methyl]-hexanoic acid,phenylmethyl ester, 3-(azidomethyl)-5-methyl-hexanoic acid,2-(2-methylpropyl)-1,4-butanedioic acid, 4-(1,1-dimethylethyl) ester,3-(azidomethyl)-5-methyl-, 1,1-dimethylethyl ester,3-(hydroxymethyl)-5-methyl-hexanoic acid, 1,1-dimethyl ester,5-methyl-3-[[[(4-methyl(phenyl)sulfonyl]oxy]-methyl-hexanoic acid,1,1-dimethylethyl ester, or4-methyl-(2-methylpropyl)-2-dioxo-5-phenyl-3-oxazolidinebutanoic acid,1,1-dimethylethyl ester.

Instead of an anti-inflammatory/anti-pain agent, the first component ofthe combination of the present invention with the hydrogen-generatingcompound can alternatively be any drug that produces inflammation as anunwanted side effect. The hydrogen-generating compound reduces theamount of inflammation caused by the drug and eliminates the unwantedside effects. It should be understood that anywhere thatanti-inflammatory/anti-pain agent is used, the drug that producesinflammation as an unwanted side effect can be used instead whereappropriate. Statins (HMG-CoA reductase inhibitors) are one type of drugthat produces an unwanted side effect of inflammation (such as myositis,myalgia, and rhabdomyolysis). Statins are generally indicated forlowering cholesterol levels in blood and for preventing heart attacksand stroke. Different statins include atorvastatin, fluvastain,lovastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, andstatin combinations with other agents.

One particular statin that can be used with the hydrogen-generatingcompound is atorvastatin (LIPITOR®, Pfizer). Atorvastatin is also knownas(3R,5R)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoate,and is described in U.S. Pat. Nos. 4,681,893; 5,273,995; 5,686,104;6,126,971; and 5,969,156. Atorvastatin calcium is shown below in FormulaV:

Atorvastatin can be administered in oral form in doses of 10 to 80mg/day; however, due to synergism with the hydrogen-generating compound,a lower dose can be preferred and normal side effects experienced can bereduced or eliminated. Any other administration methods as describedherein can also be used.

The second component of the combination of the present invention is thehydrogen-generating compound. This compound can act to generate anincrease of hydrogen in the body by several different methods, such as,but not limited to, releasing hydrogen in the body, or inducingproduction of hydrogen in the body. The generation of hydrogen can occuranywhere in the body as desired, and can be tailored to occur in aspecific site. The hydrogen-generating compound can be H2 itself, or acomposition that includes a releasable H2 moiety. Alternatively, thehydrogen-generating compound can be a compound that induces hydrogen tobe released by the body itself or by another compound in the body. Forexample, the hydrogen-generating compound can be any compound thatinduces bacteria in the stomach to release H2. The hydrogen-generatingcompound can also be H2 infused liquid that can be a drink oradministered by intravenous infusion, or any other method describedherein. Combinations of any of the methods of generating hydrogen in thebody can also be used.

The hydrogen-generating compound can be a sugar that induces hydrogenproduction in the stomach. Most preferably, the hydrogen-generatingcompound is lactulose. The hydrogen-generating compound can also behomologues of lactulose, or other sugars and their homologues. Lactuloseis a synthetic sugar that is a disaccharide of a molecule of fructoseand a molecule of galactose. It is currently indicated for constipationtreatment or for hepatic encephalopathy in removing ammonia from blood.Lactulose is non-absorbable and fermented by intestinal bacteria,resulting in the production of hydrogen. A lactulose hydrogen breathtest has previously been used to detect irritable bowel syndrome bydetecting an abnormal amount of hydrogen in the breath. However, thecreation of hydrogen in the present invention is a positive effect ofthe compound so that inflammation can be treated. Lactulose can beadministered in doses from 40 mL to over 1000 mL per day; however, dueto the synergism with the anti-inflammatory/anti-pain agent, a lowerdose can be preferred.

Alternatively, the hydrogen-generating compound can be any othermonosaccharide, polysaccharide, or other non-saccharide sweetener, suchas, but not limited to, glucose, galactose, fructose, mannitol, inulin,sucralose, aspartame, dextrose, maltodextrin, or combinations thereof.One of the preferred hydrogen-generating compounds is inulin, discussedin Example 2.

The hydrogen-generating compound can be delivered at the same time asthe anti-inflammatory/anti-pain agent, or at different times. Thehydrogen-generating compound can be contained within its own dosageform, within the dosage form together with theanti-inflammatory/anti-pain agent (i.e. a capsule containing thehydrogen-generating compound and the anti-inflammatory/anti-pain agent),or within the dosage form itself (i.e. a capsule coating that includesthe hydrogen-generating compound, with the anti-inflammatory/anti-painagent within the capsule). The composition of the present invention canbe tailored to provide different release profiles as needed or desiredfor a particular patient, such as, but not limited to, sustainedrelease, prolonged release, or immediate release. Thehydrogen-generating compound and the anti-inflammatory/anti-pain agentcan each have the same release profiles or different release profiles.

Some of the more preferred combinations of the composition of thepresent invention include pregabalin and lactulose, ibuprofen andlactulose, acetaminophen and lactulose, rofecoxib and lactulose, andcelecoxib and lactulose.

The anti-inflammatory/anti-pain agent and hydrogen-generating compoundact in a synergistic manner. Therefore, the anti-inflammatory/anti-painagent is preferably present in an amount that is lower than the normaleffective dose. The hydrogen-generating compound can also be present inan amount that is lower than the normal effective dose. In other words,by combining the anti-inflammatory/anti-pain agent with thehydrogen-generating compound, the effective amount needed can bereduced, which in turn reduces unwanted side effects. Therefore, theanti-inflammatory/anti-pain agent and the hydrogen-generating compoundcan be present in synergistically effective amounts. This combinationalso allows for the use of anti-inflammatory/anti-pain agents that havepreviously been thought to be too toxic. As shown below in Example 1,hydrogen generated by the hydrogen-generating compound lactulose showedan additive effect on pregabalin-mediated suppression of pain associatedfactors (Substance P and nerve growth factor (NGF)) in mice.

The mechanism of action of the present invention is as follows. Thehydrogen that is generated from the hydrogen-generating compound affectsthe total positive/negative charge of neuron cell surfaces to increasetheir sensitivity to the anti-inflammatory/anti-pain agent. Withpregabalin in particular, the anti-pain effect is derived from itsblocking of voltage gated calcium channels expressed on neuron cells.These channels' expression level is dependent on the total charge of theneuron cell surface. Other mechanisms of action can alternatively oradditionally be present. For example, the hydrogen-generating compoundin combination with the anti-inflammatory/anti-pain agent also reducesexcessive levels of NGF and Substance P that are present due toinflammation. The combination also reduces or down-regulates expressionof IL-1β, TNF-α, and PGE2, all mediators of inflammation and pain.

The composition of the present invention can be used for treating manydifferent diseases and conditions in which inflammation and/or pain areassociated. These diseases and conditions can be, but are not limitedto, acne vulgaris, asthma, autoimmune diseases, celiac disease, chronicprostatitis, glomerulonephritis, hypersensitivities, inflammatory boweldiseases, pelvic inflammatory disease, reperfusion injury, rheumatoidarthritis, sarcoidosis, transplant rejection, vasculitis, interstitialcystitis, atherosclerosis, allergies, myopathies, leukocyte defects,cancer, endometriosis, and multiple sclerosis. Any of these diseases canbe treated according to the methods detailed below.

The present invention provides for a method of treating inflammationand/or pain, by administering an effective amount of the compositionincluding the anti-inflammatory/anti-pain agent and thehydrogen-generating compound to an individual. Theanti-inflammatory/anti-pain agent can be any of those described above,and the hydrogen-generating compound can be any of those describedabove. Administration can be oral, by injection, topical, or any otheradministration profile described herein. The anti-inflammatory/anti-painagent and the hydrogen-generating compound preferably actsynergistically to treat inflammation and/or pain. By performing thismethod, side effects normally experienced by patients who areadministered anti-inflammatory/anti-pain agents can be reduced byreducing the dose needed due to be effective. The method can furtherinclude the step of increasing neuron cell surfaces sensitivity to theanti-inflammatory/anti-pain agent. When the anti-inflammatory/anti-painagent is pregabalin, the method can also include the step of blockingvoltage gated calcium channels expressed on neuron cells. A medicalpractitioner can confirm that inflammation has been treated in a patientby measuring an amount of IL-1β, which is a marker for inflammation, orby detecting other markers known in the art. This method can also beperformed using a drug that produces inflammation as an unwanted sideeffect in combination with the hydrogen-generating compound.

The present invention also provides for a method of treating pain, byadministering an effective amount of the composition including theanti-inflammatory/anti-pain agent and the hydrogen-generating compound.The anti-inflammatory/anti-pain agent can be any of those describedabove, and the hydrogen-generating compound can be any of thosedescribed above. Administration can be oral, by injection, topical, orany other administration profile described herein. Theanti-inflammatory/anti-pain agent and the hydrogen-generating compoundpreferably act synergistically to treat pain. By performing this method,side effects normally experienced by patients who are administeredanti-inflammatory/anti-pain agents can be reduced by reducing the doseneeded due to be effective. The method can further include the step ofincreasing neuron cell surfaces sensitivity to theanti-inflammatory/anti-pain agent. When the anti-inflammatory/anti-painagent is pregabalin, the method can also include the step of blockingvoltage gated calcium channels expressed on neuron cells. A medicalpractitioner can confirm that pain has been treated in a patient bymeasuring an amount of nerve growth factor (NGF) or Substance P, whichare markers for pain, or by detecting other markers known in the art.This method can also be performed using a drug that producesinflammation as an unwanted side effect in combination with thehydrogen-generating compound.

The present invention also provides a method of potentiating the effectsof an anti-inflammatory/anti-pain agent, by administering asynergistically effective amount of the composition including theanti-inflammatory/anti-pain agent and the hydrogen-generating compound.Most preferably, the anti-inflammatory/anti-pain agent is pregabalin andthe hydrogen-generating compound is lactulose. Alternatively, theanti-inflammatory/anti-pain agent can be any of those described above,and the hydrogen-generating compound can be any of those describedabove. Administration can be oral, by injection, topical, or any otheradministration profile described herein. Because theanti-inflammatory/anti-pain agent and the hydrogen-generating compoundinteract synergistically, the response and effect on the patient of theanti-inflammatory/anti-pain agent is greater at a particular dose whencombined with the hydrogen-generating compound than the response wouldbe alone. Therefore, by performing this method, side effects normallyexperienced by patients who are administered anti-inflammatory/anti-painagents can be reduced by reducing the dose needed due to be effective.This method can also be performed using a drug that producesinflammation as an unwanted side effect in combination with thehydrogen-generating compound.

The hydrogen-generating compound can also be used on its own, withoutthe anti-inflammatory/anti-pain agent to reduce inflammation byincreasing the amount of hydrogen in the individual. Thehydrogen-generating compound reduces inflammation by reducing excessivelevels of NGF and Substance P that are present due to inflammation, andby reducing or down-regulating expression of IL-1β, TNF-α, and PGE2. Thehydrogen-generating compound can be any hydrogen-generating compound asdescribed above. The hydrogen-generating compound can be used to reduceinflammation caused by any of the diseases described above.

The compounds of the present invention are administered and dosed inaccordance with good medical practice, taking into account the clinicalcondition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. Thepharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

In the method of the present invention, the compound of the presentinvention can be administered in various ways. It should be noted thatit can be administered as the compound and can be administered alone oras an active ingredient in combination with pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles. The compounds can beadministered orally, subcutaneously or parenterally includingintravenous, intraarterial, intramuscular, intraperitoneally,intratonsillar, and intranasal administration as well as intrathecal andinfusion techniques. Implants of the compounds are also useful. Thepatient being treated is a warm-blooded animal and, in particular,mammals including man. The pharmaceutically acceptable carriers,diluents, adjuvants and vehicles as well as implant carriers generallyrefer to inert, non-toxic solid or liquid fillers, diluents orencapsulating material not reacting with the active ingredients of theinvention.

The doses can be single doses or multiple doses over a period of severaldays. The treatment generally has a length proportional to the length ofthe disease process and drug effectiveness and the patient species beingtreated.

When administering the compound of the present invention parenterally,it will generally be formulated in a unit dosage injectable form(solution, suspension, emulsion). The pharmaceutical formulationssuitable for injection include sterile aqueous solutions or dispersionsand sterile powders for reconstitution into sterile injectable solutionsor dispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, may also be used as solvent systems for compoundcompositions. Additionally, various additives which enhance thestability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, andbuffers, can be added. Prevention of the action of microorganisms can beensured by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, for example, aluminum monostearate and gelatin.According to the present invention, however, any vehicle, diluent, oradditive used would have to be compatible with the compounds.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various of the other ingredients,as desired.

A pharmacological formulation of the present invention can beadministered to the patient in an injectable formulation containing anycompatible carrier, such as various vehicle, adjuvants, additives, anddiluents; or the compounds utilized in the present invention can beadministered parenterally to the patient in the form of slow-releasesubcutaneous implants or targeted delivery systems such as monoclonalantibodies, vectored delivery, iontophoretic, polymer matrices,liposomes, and microspheres. Examples of delivery systems useful in thepresent invention include: U.S. Pat. Nos. 5,225,182; 5,169,383;5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233;4,447,224; 4,439,196; and 4,475,196. Many other such implants, deliverysystems, and modules are well known to those skilled in the art.

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided for thepurpose of illustration only, and are not intended to be limiting unlessotherwise specified. Thus, the invention should in no way be construedas being limited to the following examples, but rather, should beconstrued to encompass any and all variations which become evident as aresult of the teaching provided herein.

Example 1

Mice were placed in the following administration groups, andadministration occurred daily:

1) Control (no treatment) (n=4)

2) Placebo (PBS, i.p.) inoculation—(i.e. CFA only) (n=4)

3) H2 enriched PBS inoculation (i.e. CFA+H2) (n=4)

4) Lactulose inoculation at 0.5 mL/mouse (i.e. CFA+Lact) (n=4)

5) LYRICA® (i.e. CFA+Lyr) (n=5)

6) LYRICA®+H2 enriched PBS (i.e. CFA+Lyr+H2) (n=5)

7) LYRICA®+Lactulose in PBS (i.e. CFA+Lact+H2) (n=4)

CFA was injected into the mice in groups 2 through 7 above. Inflammationwas induced in the mice by injection of Freund's complete adjuvant(FCA), 25 μL/foot, on day 0 as shown in FIG. 1A. Chronic inflammationoccurred until day 8 (FIG. 1B), when measurements were taken. Thesemeasurements included thickness of foot pad (level of inflammation),amount of H2 in abdominal cavity, amount of nerve growth factor (NGF)(pain marker) in foot pad, amount of Substance P (pain marker) in footpad, and amount of IL-1β in foot pad (level of inflammation).

FIG. 2 shows the results of the foot pad thickness in mm on day 7. Micein group 2 (receiving only CFA) had the largest foot pad thickness. Micein group 7 receiving LYRICA® plus lactulose had the smallest foot padthickness (besides control). While mice in group 6 (CFA+Lyr+H2) hadsmaller foot pad thickness than mice in group 5 (CFA+Lyr), mice in group7 had a significantly lower foot pad thickness than mice in group 5.Thus, LYRICA® and lactulose acted synergistically in reducing foot padthickness.

FIG. 3 shows the measurement of H2 concentration in the abdomen of themice on day 8. H2 concentration was highest with group 4, and still highwith group 7. This confirms that lactulose induces the production ofhydrogen in the abdomen.

FIG. 4 shows the measurement of the pain marker NGF in the foot pad ofmice. Large amounts of NGF were found in the mice of group 2 (CFA only).Both groups 6 and 7 of the mice showed very reduced amounts of NGF. Thisconfirms that LYRICA® and a hydrogen-generating compound (either H2enriched PBS or lactulose) can reduce pain.

FIG. 5 shows the measurement of the pain marker Substance P in thetissue of mice. Mice in group 5 showed a reduction in the markercompared to the placebo group 2. Mice in group 6 showed lower levels ofthe marker than group 5; however, mice in group 7 showed a significantlylower level of the marker than the mice in group 5. This confirms thatLYRICA® and lactulose acted synergistically in reducing the pain markerSubstance P, and thus acted synergistically in reducing pain.

FIG. 6 shows the measurement of IL-1β in tissue of the mice as anindication of the level of inflammation. Mice in group 5 had slightlyreduced levels of inflammation compared to placebo group 2. Mice ingroup 6 showed lower levels of inflammation than group 5; however, micein group 7 showed significantly lower levels of inflammation than themice in group 5. This confirms that LYRICA® and lactulose actedsynergistically in reducing levels of IL-1β, and thus actedsynergistically in reducing inflammation.

Example 2

Inulin is increasingly used in processed foods because it has unusuallyadaptable characteristics. Inulin is a starchy substance found in a widevariety of fruits, vegetables, and herbs, including wheat, garlic,onions, bananas, leeks, artichokes, and asparagus. The inulin that isused for medicine is most commonly obtained by soaking chicory roots inhot water.

Its flavor ranges from bland to subtly sweet (approx. 10% sweetness ofsugar/sucrose). It can be used to replace sugar, fat, and flour. This isadvantageous because inulin contains 25-35% of the food energy ofcarbohydrates (starch, sugar). While inulin is a versatile ingredient,it also has health benefits. Inulin increases calcium absorption andpossibly magnesium absorption. It supports the growth of a special kindof bacteria that are associated with improving bowel function andgeneral health. Inulin also decreases the body's ability to make certainkinds of fats. In terms of nutrition, it is considered a form of solublefiber and is sometimes categorized as a prebiotic. Due to the body'slimited ability to process fructans, inulin has minimal increasingimpact on blood sugar, and—unlike fructose—is not insulemic and does notraise triglycerides.

FIG. 7 shows the increased H2 in mouse abdominal cavities afteringestion of various sugar solutions. Lactose, glucose, galactose,fructose, mannitol, inulin, Splenda® (McNeil Nutritionals (sucralose)),Equal® (Merisant (aspartame, dextrose, and maltodextrin)), and lactulosewere tested along with a control. Lactulose provided the highestconcentration of H2 in ascites fluid and inulin provided the nexthighest concentration.

FIG. 8 shows a comparison of H2-enriched water versus lactulose on theconcentration of H2 in ascites fluid. After 30 minutes, H2-enrichedwater provided approximately 40 μM H2 concentration but dropped off toapproximately 15 μM after 4 hours. At 4 hours, lactulose provided over20 μM H2 concentration.

FIGS. 9A and 9B show the chemical structure of inulin and lactulose.Similar structure of the two compounds is boxed.

Example 3

The following is an in vitro experiment to test the effects of hydrogenon the Celecoxib- or Ibuprofen-mediated anti-inflammatory activities,and the results are shown in FIGS. 10 and 11.

The mouse macrophage cell line (RAW264.7) was seeded at a density of1.0×10⁵ cells/well in 96-well culture plates in Minimum Essential MediumAlpha (−MEM, Sigma) supplemented with penicillin G solution (100 U/ml,GIBCO; Invitrogen, Buffalo, N.Y.), streptomycin (100 g/ml, GIBCO;Invitrogen), and gentamicin (50 g/ml, GIBCO; Invitrogen) containing 10%FBS in the presence or absence of H₂ (300 nM/ml) for 30 min. Then thecells were exposed to LPS (0.1 mg/ml, Invivogen, San Diego, Calif.),with or without a variety of concentrations of Celecoxib or Ibuprofen(10-1000 nM). After culture of the RAW264.7 cells for 24 hours in a CO₂incubator (37° C.), the culture supernatant was harvested and subjectedto TNF-α ELISA (R&D Systems).

As shown in FIG. 10, H2 in combination with Celecoxib greatly reducedthe amount of TNF-α production in the macrophages as compared toCelecoxib administered alone, especially at the 1 μM dose. Also, asshown in FIG. 11, H2 in combination with Ibuprofen reduced TNF-αproduction in the macrophages as compared to Ibuprofen administeredalone, especially at the 0.1 dose. Therefore, H2 administered witheither Celecoxib or Ibuprofen is capable of reducing inflammation in agreater amount than the Celecoxib or Ibuprofen alone.

Example 4

The following experiment was performed to test the effects of Lactulosecombined with Celecoxib or Ibuprofen on Freund's completeadjuvant-induced hind paw inflammation in mice, with the results shownin FIGS. 12-15.

For induction of hindpaw inflammation, mice (BALB/c; 8 w old malen=6/group) received i.pl. injection of 25 μl of complete Freund'sadjuvant (CFA, diluted 1:1 with PBS, 2 mg/ml; Mycobacteriumtuberculosis; Difco Laboratories, Detroit, Mich.). Lactulose (10% water)or control water alone was administered once in a day for 7 days (500μl/day) by oral gavage using a 20-G bulb-tipped gastric gavage needle(P.O.). Celecoxib (Toronto Research Chemicals) and Ibuprofen (Enzo LifeSciences) dissolved in PBS was applied to mice once a day viaintra-peritoneal (i.p.) injection (10 mg/kg/day, respectively) for 7days. The thickness of the foot pad was measured using a digitalmicrocaliper as mice were sacrificed on 7 day. Surgically removed hindpaw tissue was homogenized in EIA-Buffer (Cayman) and subjected to mouseNGF ELISA (Millipore), PGE2 EIA (Cayman), or TNF-α ELISA (R&D systems).

As shown in FIG. 12, foot pad swelling was reduced in mice givenCFA+lactulose, CFA+Celecoxib+lactulose, and CFA+Ibuprofen+lactulose, ascompared to mice who did not receive lactulose. FIG. 13 shows that theamount of PGE2 was reduced in mice given CFA+lactulose,CFA+Celecoxib+lactulose, and CFA+Ibuprofen+lactulose, as compared tomice who did not receive lactulose. FIG. 14 shows that NGF was reducedin mice given CFA+lactulose, and CFA+Ibuprofen+lactulose, as compared tomice who did not receive lactulose. FIG. 15 shows that the amount ofTNF-α was greatly reduced in mice given CFA+lactulose,CFA+Celecoxib+lactulose, and CFA+Ibuprofen+lactulose, as compared tomice who did not receive lactulose. Therefore, lactulose plus eitherCelecoxib or Ibuprofen is capable of reducing inflammation in a greateramount than the Celecoxib or Ibuprofen alone.

Throughout this application, various publications, including UnitedStates patents, are referenced by author and year and patents by number.Full citations for the publications are listed below. The disclosures ofthese publications and patents in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology, which has been used is intended tobe in the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims, the inventioncan be practiced otherwise than as specifically described.

1. A composition for treating inflammation and pain, comprisingsynergistic amounts of an anti-inflammatory agent or anti-pain agent incombination with a hydrogen-generating compound.
 2. The composition ofclaim 1, wherein said anti-inflammatory or anti-pain agent is chosenfrom the group consisting of non-steroidal anti-inflammatory drugs(NSAIDS), steroids, immune selective anti-inflammatory derivatives(ImSAIDs), narcotics, analgesics, biologic agents, and combinationsthereof.
 3. The composition of claim 2, wherein said NSAID is ibuprofen.4. The composition of claim 3, wherein said ibuprofen is in an amount of200 to 300 mg per day.
 5. The composition of claim 2, wherein said NSAIDis celecoxib.
 6. The composition of claim 5, wherein said celecoxib isin an amount of 50 to 400 mg per day.
 7. The composition of claim 2,wherein said NSAID is rofecoxib.
 8. The composition of claim 7, whereinsaid rofecoxib is in an amount of 12.5 to 50 mg per day.
 9. Thecomposition of claim 1, wherein said anti-inflammatory or anti-painagent is pregabalin.
 10. The composition of claim 9, wherein saidpregabalin is in an amount of 25 to 600 mg per day.
 11. The compositionof claim 9, wherein said pregabalin is chosen from the group consistingof the S-enantiomer of 3-(aminomethyl)-5-methyl-hexanoic acid,(S)-(+)-enantiomer of 4-amino-3-(2-methylpropyl)butanoic acid, and the(R)(−)-enantiomer of 4-amino-3-(2-methylpropyl)butanoic acid.
 12. Thecomposition of claim 1, wherein said hydrogen-generating compound ischosen from the group consisting of H₂ molecules, a prodrug releasingH₂, a compound that causes the release of H₂ within the body, andcombinations thereof.
 13. The composition of claim 1, wherein saidhydrogen-generating compound is a compound that induces bacteria in thestomach to release H₂.
 14. The composition of claim 1, wherein saidhydrogen-generating compound is an H₂ infused liquid.
 15. Thecomposition of claim 1, wherein said hydrogen-generating compound is asugar that induces hydrogen production in the stomach chosen from thegroup consisting of lactulose, glucose, galactose, fructose, mannitol,inulin, sucralose, aspartame, dextrose, maltodextrin, and combinationsthereof.
 16. The composition of claim 15, wherein said sugar islactulose and is in an amount of 40 mL to 1000 mL per day.
 17. Thecomposition of claim 1, wherein said composition is in a dosage form ischosen from the group consisting of the hydrogen-generating compound inits own dosage form, the hydrogen-generating compound and theanti-inflammatory or anti-pain agent in the same dosage form, or thehydrogen-generating compound within a coating of the dosage form and theanti-inflammatory or anti-pain agent within the dosage form.
 18. Thecomposition of claim 1, wherein said composition is chosen from thegroup consisting of pregabalin and lactulose, ibuprofen and lactulose,acetaminophen and lactulose, rofecoxib and lactulose, and celecoxib andlactulose.
 19. A composition for treating inflammation and pain,comprising synergistically effective amounts of pregabalin andlactulose. 20-70. (canceled)